High-Level Heterologous Expression of Bacillus halodurans Putative Xylanase Xyn11A (BH0899) in Kluyveromyces lactis

The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 μg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or β-xylosidase activity.     

Xyn11E from Paenibacillus barcinonensis BP-23: a LppX-chaperone-dependent xylanase with potential for upgrading paper pulps

A new xylanase from Paenibacillus barcinonensis BP-23, Xyn11E, has been identified and characterized. Xyn11E has been cloned and heterologously expressed in Escherichia coli. It is a single-domain xylanase belonging to the family 11 of glycosyl hydrolases (GH11) with a predicted molecular weight of 20.652 kDa and an isoelectric point (pI) of 8.7. Substrate specificity, kinetic properties, and mode of action of the purified xylanase were characterized. Xyn11E exhibited high activity toward branched xylans, being beechwood xylan the preferred substrate. The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. Catalytic constants were determined on beechwood xylan, on which Xyn11E showed a Km of 12.98 mg/ml and a Vmax of 3,023 U/mg. The enzyme hydrolyzed long xylooligosaccharides, while oligomers shorter than xylotetraose were not degraded. Products released from glucuronoxylans were shorter than those liberated from cereal arabinoxylans. The xylanase was dependent on P. barcinonensis BP-23 LppX for its expression in an active form. Coexpression of Xyn11E with E. coli chaperones could not replace the need of LppX, which seems to act as a specific chaperone for Xyn11E correct folding. Activity of the enzyme on bleached pulps was evaluated. Xyn11E liberated reducing sugars from ECF and TCF pulps from eucalyptus, sisal, and flax, which makes it a good candidate for the enzymatic-assisted production of high-cellulose-content pulps from paper-grade pulps.     

Activity and thermostability increase of xylanase following transplantation with modules sub-divided from hyper-thermophilic CBM9_1-2

Transplantation is useful for elucidating the functions of structural modules and for engineering enzyme properties. Unexpectedly, transplanting a hyper-thermophilic carbohydrate-binding module, CBM9_1-2, into the mesophilic Aspergillus niger GH11 xylanase (Xyn) slightly decreased the thermal inactivation half-life of Xyn. This effect was further investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras Xyn-C1 and Xyn-C2. Both chimeras exhibited higher catalytic activities on xylan than native Xyn. Xyn-C2 exhibited higher binding affinities for both oat spelt and birch wood xylans, and its thermal inactivation half-life (69.3 min) was 4 or 5 times longer than that of Xyn (17.6 min), Xyn-C1 (13.4 min), and the original chimera containing CBM9_1-2 (13.8 min). In contrast, Xyn-C1 exhibited higher binding affinity for oat spelt xylan, but not for birch wood xylan. Through this rational engineering of the fungal xylanase, the C2 sub-module was shown to have a different thermostabilizing effect than the C1 sub-module. The different functions of the smaller parts of a large module can play pivotal roles in transplantation.     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998     

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.     

Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Paenibacillus curdlanolyticus B-6 showed effective degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approximately 1,450 kDa) containing several xylanases and cellulases. To characterize the multienzyme complex, we purified the complex from culture supernatants by four kind of chromatography. The purified multienzyme complex was composed of a 280-kDa protein with xylanase activity, a 260-kDa protein that was a truncated form on the C-terminal side of the 280-kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and carboxymethyl cellulase activities. The 280-kDa protein resembled the scaffolding proteins of cellulosomes based on its migratory behavior in polyacrylamide gels and as a glycoprotein. Cloning of the 40-kDa major xylanase subunit named Xyn11A revealed that Xyn11A contained two functional domains which belonged to glycosyl hydrolase family-11 and to carbohydrate-binding module family-36, respectively, and a glycine- and asparagine-rich linker. However, an amino acid sequence similar to a dockerin domain, which is crucial to cellulosome assembly, was not found in Xyn11A. These results suggest that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin-dockerin interactions known in cellulosomes.     

<Emphasis Type="Italic">C</Emphasis>-Terminal carbohydrate-binding module 9_2 fused to the <Emphasis Type="Italic">N</Emphasis>-terminus of GH11 xylanase from <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Objective

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module.

Results

A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn.

Conclusion

C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

     

Fusion of the OsmC domain from esterase EstO confers thermolability to the cold-active xylanase Xyn8 from <Emphasis Type="Italic">Pseudoalteromonas arctica</Emphasis>

The OsmC-region (osmotically induced protein family) of the two-domain esterase EstO from the psychrotolerant bacterium Pseudoalteromonas arctica has been shown to increase thermolability. In an attempt to test if these properties can be conferred to another enzyme, we genetically fused osmC to the 3′-region of the family 8 xylanase encoding gene xyn8 from P. arctica. The chimeric open reading frame xyn8-OsmC was cloned and the chimeric protein was purified after heterologous expression in Escherichia coli. Xyn8 and Xyn8-OsmC showed cold-adapted properties (more than 60% activity at 0°C) using birchwood xylan as the preferred substrate. Maximal catalytic activity is slightly shifted from 15°C (Xyn8) to 20°C for Xyn8-OsmC. Thermostability of Xyn8-OsmC is significantly changed in comparison to wild-type Xyn8. The OsmC-fusion variant showed an apparent decrease in thermostability between 40 and 45°C, while both proteins are highly instable at 50°C.     

Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris

Novel xylanase (EC 3.2.1.8) is in great demand due to its industrial significance. In this study, we have developed and characterized a novel xylanase-producing yeast strain. This mature xylanase gene xyn11A consists of 870 base pairs and belongs to GH11 family. The gene sequence was optimized and synthesized, and was then cloned into yeast vector pGAPZαA under the control of the constitutive GAP promoter. SDS-PAGE analysis indicates that Xyn11A is extracellularly expressed as a glycosylated protein in P. pastoris. Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase retained more than 90% of its activity after incubation at 50 °C and 60 °C for up to 1 h. Xyn11A is also stable over a wide range of pH (2.0–11.0). Most metal ions tested such as copper (Cu²⁺) and lead (Pb²⁺) have little inhibitory effects on Xyn11A. It is also resistant to pepsin and proteinase K digestion, retaining 80% and 90% of its activity after digestion at 37 °C for 1 h, respectively. Those superior properties make Xyn11A a robust xylanase with great potential for industrial use. To the best of our knowledge, this is the first report of xylanase from the fungus Corynascus thermophilus.     

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.     

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

Background

In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11 (GH10 and GH11, respectively) xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates.

Results

The xylanases from Nonomuraea flexuosa (Nf Xyn11A) and from Thermoascus aurantiacus (Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively.

Conclusions

Because of its high catalytic activity and good thermostability, T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas N. flexuosa xylanase shows more significant potential for the production of XOSs.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

High-level heterologous expression of Bacillus halodurans putative xylanase xyn11a (BH0899) in Kluyveromyces lactis

The putative xyn11A structural gene (BH0899) encoding a family-11 xylanase from alkaliphilic Bacillus halodurans strain C-125 was heterologously expressed in the yeast Kluyveromyces lactis CBS 1065 and secreted to a level of 156 microg/ml under selective culture conditions in shake flasks. The Xyn11A production level in shake flask cultures of K. lactis CBS 1065 was higher than that reported for other xylanase genes placed under the control of the regulated LAC4 promoter on a plasmid containing an entire sequence of pKD1 from Kluyveromyces drosophilarium. Recombinant Xyn11A was highly active over pH range from 3 to 10, with maximal activity around pH 7. The enzyme showed a specific activity of 628 U/mg-protein on birchwood xylan as substrate, but no cellulase or beta-xylosidase activity.     

Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824

A thermostable xylanase gene, xyn10A (CAP0053), was cloned from Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the C. acetobutylicum xyn10A gene encoded a 318-amino-acid, single-domain, family 10 xylanase, Xyn10A, with a molecular mass of 34 kDa. Xyn10A exhibited extremely high (92%) amino acid sequence identity with Xyn10B (CAP0116) of this strain and had 42% and 32% identity with the catalytic domains of Rhodothermus marinus xylanase I and Thermoascus aurantiacus xylanase I, respectively. Xyn10A enzyme was purified from recombinant Escherichia coli and was highly active toward oat-spelt and Birchwood xylan and slightly active toward carboxymethyl cellulose, arabinogalactouronic acid, and various p-nitrophenyl monosaccharides. Xyn10A hydrolyzed xylan and xylooligosaccharides larger than xylobiose to produce xylose. This enzyme was optimally active at 60°C and had an optimum pH of 5.0. This is one of a number of related activities encoded on the large plasmid in this strain.     

A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428

A cellulase free thermostable xylanase from Streptomyces sp. CS428 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular xylanase was purified 26 fold with a 55% yield by CM Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428) was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (4 to ～13.6) and to 50 °C and have an optimum temperature of 80 °C. Xyn428 had K_m and V_max values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM, and produced xylose and xylobiose as major products. It was found to degrade agro waste materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being simple in purification, thermo tolerant, pH stability in broad range and ability to produce xylooligosaccharides show that Xyn428 has potential applications in industries as a biobleaching agent and for xylooligosaccharides production.     

Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases

Endo-β1,4-xylanases (xylanases) hydrolyse the β1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown. Furthermore, the mechanism for the differences in the inhibition of fungal GH11 enzymes by the wheat protein XIP-I remains opaque. To address these issues we report the crystal structure and biochemical properties of the Neocallimastix patriciarum xylanase NpXyn11A, which displays unusually high catalytic activity and is one of the few fungal GH11 proteins not inhibited by XIP-I. Although the structure of NpXyn11A could not be determined in complex with substrates, we have been able to investigate how GH11 enzymes hydrolyse decorated substrates by solving the crystal structure of a second GH11 xylanase, EnXyn11A (encoded by an environmental DNA sample), bound to ferulic acid-1,5-arabinofuranose-α1,3-xylotriose (FAX₃). The crystal structure of the EnXyn11A-FAX₃ complex shows that solvent exposure of the backbone xylose O2 and O3 groups at subsites −3 and +2 allow accommodation of α1,2-linked 4-methyl-D-glucuronic acid and L-arabinofuranose side chains. Furthermore, the ferulated arabinofuranose side chain makes hydrogen bonds and hydrophobic interactions at the +2 subsite, indicating that the decoration may represent a specificity determinant at this aglycone subsite. The structure of NpXyn11A reveals potential −3 and +3 subsites that are kinetically significant. The extended substrate-binding cleft of NpXyn11A, compared to other GH11 xylanases, may explain why the Neocallimastix enzyme displays unusually high catalytic activity. Finally, the crystal structure of NpXyn11A shows that the resistance of the enzyme to XIP-I is not due solely to insertions in the loop connecting β strands 11 and 12, as suggested previously, but is highly complex.     

The phytotoxic activity of the cerato‐platanin BcSpl1 resides in a two‐peptide motif on the protein surface

Cerato‐platanin family proteins are secreted and have been found in both the fungal cell wall and the extracellular medium. They elicit defence responses in a variety of plants and have been proposed to be perceived as pathogen‐associated molecular patterns (PAMPs) by the plant immune system, although, in the case of the necrotroph Botrytis cinerea, the cerato‐platanin BcSpl1 contributes to fungal virulence instead of plant resistance. In this study, we report that BcSpl1, which was previously found in the secretome as an abundant protein, is even more abundant in the fungal cell wall. By fusion to green fluorescent protein (GFP), we also show that BcSpl1 associates with the plant plasma membrane causing rapid morphological changes at the cellular level, such as the disorganization of chloroplasts, prior to macroscopic necrosis in the treated tissue. By a combination of serial deletion studies, synthetic peptides and chimeric proteins, we mapped the eliciting activity to a two‐peptide motif in the protein surface. The expression of a chimeric protein displaying this motif in B. cinerea mutants lacking BcSpl1 undoubtedly showed that the motif is responsible for the contribution of BcSpl1 to virulence.